Methods and kits for synthesis of siRNA expression cassettes

ABSTRACT

Amplification-based methods and kits for rapidly producing siRNA expression cassettes are provided. Also provided are methods for expressing amplified siRNA expression cassettes in cells.

[0001] This application claims priority to co-pending U.S. ProvisionalApplication Serial No. 60/399,718 filed Aug. 1, 2002 and 60/408,298filed Sep. 6, 2002.

GOVERNMENT RIGHTS STATEMENT

[0002] This invention was made with federal government support from theNational Institutes of Health of the U.S. Department of Health and HumanServices under Grant No. AI29329 to the City of Hope Cancer Center. TheUnited States government may have certain rights in this invention.

FIELD OF THE INVENTION

[0003] The present invention relates to RNA interference (RNAi), and isuseful for screening multiple RNAi gene constructs to identify thosemost effective against a given target.

BACKGROUND OF THE INVENTION

[0004] RNA interference (RNAi) is a process in which double stranded RNA(ds RNA) induces the postranscriptional degradation of homologoustranscripts, and has been observed in a variety of organisms includingplants, fungi, insects, protozans, and mammals. (Moss, E. G., et al.,2001; Bernstein, E., et al., 2001; Elbashir, S. M., et al., 2001;Elbashir, S. M., et al., 2001). RNAi is initiated by exposing cells todsRNA either via transfection or endogenous expression. Double-strandedRNAs are processed into 21 to 23 nucleotide (nt) fragments known assiRNA (small interfering RNAs). (Elbashir, S. M., et al., 2001;Elbashir, S. M., et al., 2001). These siRNAs form a complex known as theRNA Induced Silencing Complex or RISC (Bernstein, E., et al., Hammond,S. M., et al. 2001), which functions in homologous target RNAdestruction. In mammalian systems, the sequence specific RNAi effect canbe observed by introduction of siRNAs either via transfection orendogenous expression of 21-23 base transcripts or longer hairpinprecursors. Use of siRNAs evades the dsRNA induced interferon and PKRpathways that lead to non-specific inhibition of gene expression.(Elbashir, S. M., et al., 2001).

[0005] Recently, several groups have demonstrated that siRNAs can beeffectively transcribed by Pol III promoters in human cells and elicittarget specific mRNA degradation. (Lee, N. S., et al., 2002; Miyagishi,M., et al., 2002; Paul, C. P., et al., 2002; Brummelkamp, T. R., et al.,2002; Ketting, R. F., et al., 2001). These siRNA encoding genes havebeen transiently transfected into human cells using plasmid or episomalviral backbones for delivery. Transient siRNA expression can be usefulfor rapid phenotypic determinations preliminary to making constructsdesigned to obtain long term siRNA expression. Of particular interest isthe fact that not all sites along a given mRNA are equally sensitive tosiRNA mediated downregulation. (Elbashir, S. M., et al., 2001; Lee, N.S., et al., 2001; Yu, J. Y., et al., 2002; Holen, T, et al., 2002).

[0006] In contrast to post-transcriptional silencing involvingdegradation of mRNA by short siRNAs, the use of long siRNAs to methylateDNA has been shown to provide an alternate means of gene silencing inplants. (Hamilton, et al.). In higher order eukaryotes, DNA ismethylated at cytosines located 5′ to guanosine in the CpG dinucleotide.This modification has important regulatory effects on gene expression,especially when involving CpG-rich areas known as CpG islands, locatedin the promoter regions of many genes. While almost all gene-associatedislands are protected from methylation on autosomal chromosomes,extensive methylation of CpG islands has been associated withtranscriptional inactivation of selected imprinted genes and genes onthe inactive X-chromosomes of females. Aberrant methylation of normallyunmethylated CpG islands has been documented as a relatively frequentevent in immortalized and transformed cells and has been associated withtranscriptional inactivation of defined tumor suppressor genes in humancancers. In this last situation, promoter region hypermethylation standsas an alternative to coding region mutations in eliminating tumorsuppression gene function. (Herman, et al.). The use of siRNAs fordirecting methylation of a target gene is described in U.S. ProvisionalApplication No. 60/447,013, filed Feb. 13, 2003, which is incorporatedherein by reference.

[0007] There are at this time no rules governing siRNA target siteselection for a given mRNA target. It is therefore important to be ableto rapidly screen potential target sequences to identify a sequence orsequences susceptible to siRNA mediated degradation. Initial attempts ataddressing this problem have taken advantage of anoligonucleotide/RNAseH procedure in cell extracts on native mRNAtranscripts designed to identify sites that are accessible tobase-paring, including pairing by nucleic acid products such asribozymes. This approach has also been applied to identifying bindingsites for siRNA (Lee, N. S. et al. 2001). Having identified anaccessible site with the oligonucleotide/RNAseH procedure it is stillnecessary to generate siRNAs against the target at the accessible site.This approach has been applied to siRNA site accessibility as well.(Lee, N. S., et al., 2001). However, this process can be time consuming,and in the end it is still necessary to synthesize the siRNA genes forfinal testing.

[0008] Thus, an object of the present invention is to provide anamplification-based approach in the form of a method and kit for rapidlysynthesizing siRNA genes, so as to permit rapid screening of potentialtarget sequences susceptible to siRNA mediated degradation.

[0009] Another object of the invention is to provide a method forcontrolling or inhibiting expression of a target gene by transfecting acell with an amplified siRNA expression cassette.

SUMMARY OF THE INVENTION

[0010] The present invention provides an amplification-based approach(e.g., Polymerase Chain Reaction (PCR)) for rapid synthesis ofpromoter-containing siRNA expression cassettes, and their subsequenttransfection into cells. This approach, which includes methods and kitsfor performing the methods, can be utilized for the facile screening ofsiRNA encoding genes to identify those encoding siRNAs having the bestfunctional activity for a given target. The approach can be utilizedwith siRNAs expressed independently from promoters or with siRNAsexpressed as hairpin precursors or other precursors. The amplificationproducts produced using the approach may be used directly, withoutsubsequent cloning, by transfecting them into cells followed byfunctional assays.

[0011] The method of the present invention is fast and inexpensive,allowing multiple different siRNA gene candidates and/or promotercandidates to be rapidly screened for efficacy before cloning into avector.

[0012] The method of the present invention is useful for screening siRNAgene libraries.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1 is a schematic representation of a PCR strategy used toyield U6 transcription cassettes expressing siRNAs. The 5′ PCR primer iscomplementary to the 5′ end of the U6 promoter and is standard for allPCR reactions. A) The 3′ PCR primer is complementary to sequences at the3′ end of the U6 promoter and is followed by either the sense orantisense sequences, a stretch of four to six deoxyadenosines (Ter) andan additional “stuffer-Tag” sequence. The adenosines are the terminationsignal for the U6 Pol III promoter; therefore, any sequence added afterthis signal will not be transcribed by the Pol III polymerase and willnot be part of the siRNA. B) The sense and antisense sequences arelinked by a 9 nt loop and are inserted in the cassette by a two-step PCRreaction. C) The sense and antisense sequences linked by a 9-nucleotideloop and followed by the stretch of adenosines and by the Tag sequencesare included in a single 3′ primer. D) Complete PCR expression cassetteobtained by the PCR reaction. To amplify and identify functional siRNAsfrom the transfected cells, or to increase the yield of the PCR productshown in D, a nested PCR can be performed using the universal 5′ U6primer and a 3′ primer complementary to the Tag sequence (alsostandard), as indicated in the figure.

[0014]FIG. 2 shows the inhibition of enhanced green fluorescent protein(EGFP) expression using siRNA-containing PCR cassettes transfected in293 cells. The PCR cassettes containing either the sense, antisense, orboth sense and antisense siRNAs were co-transfected with the targetconstruct into 293 cells expressing the Ecdysone trans-activator. Thehuman immunodeficiency virus (HIV) rev target is fused to the greenfluorescent protein mRNA which is expressed from an inducible promoter.After adding Ponasterone A, EGFP expression can be detected in thecontrol cells (A), but not in cells transfected with either a mixture ofsense and antisense siRNA expressing PCR products (D), or with the PCRcassette expressing the hairpin construct (E). Panels B and C depictco-transfection of cells with target and PCR cassettes expressing sensealone (B) or antisense alone (C). The Rev-EGFP protein is primarily inthe cell nucleolus as a consequence of the nucleolar localizing signalin the Rev portion.

[0015]FIG. 3 shows the detection of siRNAs and PCR amplification ofsiRNA encoding DNAs in transfected cells. A. Northern gel analyses ofsiRNAs expressed from PCR products transfected in A293 cells. Lane 1,cells transfected with the EFGP target construct alone; Lane 2, cellstransfected with antisense encoding construct alone; Lane 3, cellsco-transfected with antisense and sense encoding constructs; Lane 4,cells transfected with hairpin expression construct. The probe iscomplementary to the antisense. In Lanes 24, the siRNA encoding DNAconstructs were co-transfected with the inducible EGFP construct. Thehairpin product appears smaller than the individually expressed siRNAs,demonstrating processing of the hairpin loop. B and C. PCR amplificationof transfected PCR constructs. B. PCR amplification of non-specificsiRNA encoding DNA from fluorescence activated cell sorting (FACS)sorted EFGP positive and negative cells. The non-functional construct isdetected in all cell fractions. Lanes 1 and 4 show the amplificationresults from the EGFP positive fractions. Lanes 2 and 3 show theamplification results from the EGFP negative fractions. C. PCRamplification of functional hairpin expression construct from FACSsorted, EGFP expressing and non-expressing cells. The amplificationresults show the presence of the functional siRNA only in the EGFPnegative fractions (lanes 2-3). In lane 4, there is a small amount ofamplified product, perhaps derived from some contaminating of EGFPnegative cells. NC indicates negative PCR controls.

[0016]FIG. 4 is a graph showing a comparison of HIV inhibition by shRNAsexpressed from a PCR product and plasmids.

[0017]FIG. 5 is a graph showing the persistence of HIV inhibition byshRNAs expressed from PCR products in accordance with the presentinvention.

[0018]FIG. 6 is a graph showing the results of an HIV inhibition testusing cloned U6+1 shRNA constructs.

[0019]FIG. 7 is a graph showing the results of HIV inhibition assaysusing a purified U6+1 shRNA PCR product in accordance with the presentinvention.

[0020]FIG. 8 is a graph showing the results of HIV inhibition assaysusing a purified U6+1 shRNA product in accordance with the presentinvention.

[0021]FIG. 9 shows schematically an embodiment of the present inventionin which a PCR-amplified siRNA expression cassette is cloned into acloning vector.

DETAILED DESCRIPTION OF THE INVENTION

[0022] The present invention provides an amplification-based method forproducing a promoter-containing siRNA expression cassette.

[0023] In one embodiment, the method comprises:

[0024] i) treating one strand of a double-stranded promoter sequence orconstruct, in an amplification reaction mixture, with an oligonucleotideprimer which is complementary to the 5′ end of the promoter sequence;

[0025] ii) treating the other strand of the promoter sequence orconstruct, in the amplification reaction mixture, with a secondoligonucleotide primer which is complementary to the 3′ end of thepromoter sequence, wherein the second primer comprises one or moresequences which are complementary to a sequence encoding a sense or(and/or) antisense sequence of a siRNA molecule, optionally along withone or both of a loop sequence and a terminator sequence; and

[0026] iii) treating the amplification reaction mixture of steps (i) and(ii) in an amplification reaction at a temperature for annealing andextending said primers on the promoter sequence or construct and at atemperature for denaturing the extension products to provide anamplified product comprising the promoter, one or more sequencesencoding the sense or (and/or) antisense sequence of the siRNA, and oneor both of the loop sequence and the terminator sequence.

[0027] The steps (i)-(iii) can be repeated a sufficient number of timesto amplify and detect the promoter-containing siRNA expression cassette.It is also recognized that alternatives to the loop sequence and/orterminator sequence may be utilized in the invention, which are capableof achieving the same function or purpose as the loop and terminatorsequences. It is also recognized that variations in the above steps areencompassed within the invention, in the event these variations alsoprovide an amplification-based method for producing apromoter-containing siRNA expression cassette. It is further recognizedthat the term complementary, although in a preferred embodiment refersto a perfect base-paired match between two sequences, may not requiresuch, and thus the term complementary also encompasses those sequencesnot having a perfect base-paired match but which are otherwise able toachieve the intended result of the invention.

[0028] The terms “loop sequence” and “terminator sequence” refer to thesequences corresponding to the loop and terminator elements, includingthe final sequences and any precursor sequences such as the sequencesencoding the final sequences, and any complementary sequences.

[0029] In a preferred embodiment, the method is a PCR-based method.However, it is recognized that the invention may be practiced based onother amplification methods known currently or in the future.

[0030] The promoter may be any promoter capable of transcribing an siRNAmolecule, and is preferably one that can transcribe siRNA in mammaliancells. In a preferred embodiment, the promoter is a Pol III promoter,more preferably a mammalian U6 promoter, and most preferably a human U6promoter. Other promoters, such as the H1 promoter, U1 or tRNA promoterssuch as tRNA Val, Met or Lys3 may also be useful in the presentinvention. It is also possible to use Pol II promoters such as the U 1snRNA promoter.

[0031] The terminator sequence may be any sequence encoding a functionalterminator sequence. In a preferred embodiment, the terminator sequencecomprises a sequence of deoxyadenosines, preferably about 4-6deoxyadenosines, and more preferably a sequence of 6 deoxyadenosines.

[0032] In another embodiment, the second primer may further comprise atag sequence to identify functional siRNA encoding sequences. In a morepreferred embodiment, the tag sequence may further comprise arestriction site useful for cloning.

[0033] In one embodiment, the second primer comprises a sequence that iscomplementary to a sequence encoding a sense sequence, along with aterminator sequence or loop sequence.

[0034] In another embodiment, the second primer comprises a sequencethat is complementary to a sequence encoding an antisense sequence,along with a terminator sequence or loop sequence.

[0035] In still another embodiment, the second primer comprises asequence that is complementary to a sequence encoding a sense sequenceand a sequence that is complementary to a sequence encoding an antisensesequence of said siRNA molecule, along with a terminator sequence.

[0036] In a preferred embodiment of the above embodiment, the sense andantisense sequences may be attached by a loop sequence. The loopsequence may comprise any sequence or length that allows expression of afunctional siRNA expression cassette in accordance with the invention.In a preferred embodiment, the loop sequence contains higher amounts ofuridines and guanines than other nucleotide bases. The preferred lengthof the loop sequence is about 4 to about 9 nucleotide bases, and mostpreferably about 8 or 9 nucleotide bases.

[0037] The amplified products of the present method will vary dependingon which embodiment above is selected.

[0038] The sequences or constructs encoding the sense and antisensesequences preferably contain about 19-29 nucleotides, more preferablyabout 19-23 nucleotides, and most preferably about 21 nucleotides. ThesiRNA molecules also may contain 3′ nucleotide, preferably 3′dinucleotide overhangs, including 3′UU. More generally, the RNAi orsiRNA molecules also include those known in the art.

[0039] In one embodiment, the amplified product comprises the promoterand a sequence or construct encoding either the sense or antisensesequence of the siRNA molecule. The amplified product also may containthe loop sequence or the terminator sequence.

[0040] In another embodiment, the amplified product comprises thepromoter, a sequence or construct encoding either the sense or antisensesequence of the siRNA molecule, and the terminator sequence.

[0041] In another embodiment, the amplified product comprises thepromoter, a sequence or construct encoding either the sense or antisensesequence of the siRNA molecule, and the loop sequence. In thisembodiment, the amplified product may be treated in anotheramplification reaction to provide another amplified product. This may beachieved using a third oligonucleotide primer. A portion of this thirdprimer is complementary to the loop sequence of the first amplifiedproduct. The third primer also comprises a sequence complementary to asequence encoding the antisense sequence when the first amplifiedproduct contains the sense encoding sequence, or a sequencecomplementary to a sequence encoding the sense sequence when the firstamplified product contains the antisense encoding sequence. The thirdprimer also may include a terminator sequence.

[0042] In another embodiment, the amplified product comprises thepromoter, a sequence or construct encoding the sense sequence and asequence or construct encoding the antisense sequence of the siRNAmolecule. In this embodiment, the sense and antisense encoding sequencesor constructs may be attached by a loop sequence. The amplified productalso may contain a terminator sequence.

[0043] In still another embodiment, amplified products are produced thatcomprise the promoter, a sequence or construct encoding the sensesequence and a sequence or construct encoding the antisense sequence ofthe siRNA molecule. The sense and antisense encoding sequences orconstructs may be attached by a loop sequence. The amplified productsalso may contain a terminator sequence.

[0044] In yet another embodiment, the amplified product, in anotheramplification reaction, can be treated with a fourth oligonucleotideprimer, a portion of which is complementary with the tag sequence.

[0045] In a preferred embodiment, the method may further comprise thestep of purifying the amplified promoter-containing siRNA expressioncassette. Various purification techniques are known in the art and maybe used in the present invention. Examples are described below.

[0046] In another embodiment, the amplified, and preferably purified,promoter-containing siRNA expression cassette produced according to theinvention is transfected into cells for screening. After transfection,the siRNA can be expressed to induce gene silencing.

[0047] In another embodiment, a selected and preferably purified,promoter-containing siRNA expression cassette is cloned into a selectedvector. For this embodiment, it is recognized that restriction sites canbe inserted at the ends of the siRNA expression cassette, preferablyduring production, for example, by including restriction site-encodingsequences within the primers. A schematic of this embodiment is shown inFIG. 9, as well as in U.S. Provisional Application No. 60/399,397, filedJul. 31, 2002, which is incorporated herein by reference.

[0048] In a preferred embodiment, the selected cells are mammaliancells.

[0049] In another preferred embodiment, one or more of theoligonucleotide primers are modified, preferably by phosphorylation.

[0050] In another embodiment, the method also comprises the step ofscreening for a target site on mRNA sensitive to the expressed siRNAmolecule.

[0051] In another embodiment, the method includes a positive and/ornegative control, such as a control cassette.

[0052] In another aspect, the invention provides a method for inhibitingexpression of a target gene. The method comprises transfecting a cellwith an amplified, and preferably purified, siRNA expression cassette sothat a siRNA can be expressed and inhibit the target gene. In apreferred embodiment, the cell is transfected with two or more differentsiRNA expression cassettes. In one embodiment, the different siRNAexpression cassettes contain different siRNA encoding genes, includingdifferent loop sequences, and/or different promoters.

[0053] In another aspect, the invention provides a method for modifyinggene function in mammals, for example by directing methylation of atarget gene, including a promoter region of the gene, by transfecting acell with an amplified siRNA expression cassette in accordance with theinvention.

[0054] In another aspect, the invention provides a PCR-based approach inthe form of a kit for producing a promoter-containing siRNA expressioncassette. The kit comprises a double-stranded, promoter-containingtemplate, an oligonucleotide primer complementary to the 5′ end of thepromoter-containing template, and an oligonucleotide primercomplementary to the 3′ end of the promoter-containing template. The 3′primer also comprises one or more sequences complementary to a sequenceencoding a sense or (and/or) antisense sequence of a siRNA molecule.

[0055] The 3′ primer may further comprise a loop sequence, in which casethe kit further comprises an oligonucleotide primer complementary to theloop sequence, which primer comprises a sequence complementary to asequence encoding a sense or antisense sequence of the siRNA molecule.

[0056] In one embodiment, the kit comprises a 3′ primer comprising asequence complementary to a sequence encoding a sense sequence andanother 3′ primer comprising a sequence complementary to a sequenceencoding an antisense sequence.

[0057] In another embodiment, the 3′ primer comprises a sequence that iscomplementary to a sequence encoding a sense sequence, a sequence thatis complementary to a sequence encoding an antisense sequence, and aterminator sequence. The sequences complementary to the sense andantisense encoding sequences preferably are attached by a loop sequence.

[0058] In a preferred embodiment, the oligonucleotide primers aremodified, preferably by phosphorylation.

[0059] The kit also may comprise PCR amplification reagents and reagentsfor purifying the amplified siRNA expression cassette.

[0060] In another preferred embodiment, the kit also comprises one orboth of a positive and negative control.

[0061] Preferred embodiments of the invention are described below;however, the invention is understood not to be limited to the followingembodiments.

[0062] PCR Amplification, Transfection, and Expression of siRNAs inMammalian Cells.

[0063] The procedure for a PCR-based approach is depicted schematicallyin FIG. 1. In a preferred embodiment, universal primer that iscomplementary to the 5′ end of the human U6 promoter is used in a PCRreaction along with a primer(s) complementary to the 3′ end of thepromoter, which primer harbors appended sequences which arecomplementary to the sense or antisense siRNA genes (FIG. 1A). The senseor antisense sequences are followed by a transcription terminatorsequence (Ter), which is preferably a stretch of about 4-6deoxyadenosines, and more preferably a stretch of 6 deoxyadenosines, andby a short additional “stuffer-tag” sequence that may include arestriction site for possible cloning at a later stage. The resultingPCR products include the U6 promoter sequence, the siRNA sense orantisense encoding sequence, a terminator sequence, and a short tagsequence at the 3′ terminus of the product.

[0064] In another embodiment, both the sense and antisense sequences canbe included in the same cassette (FIGS. 1B, 1D). In this case anucleotide loop, preferably containing 9 nucleotides, is insertedbetween the sense and antisense siRNA sequences. The resulting singlePCR product includes the U6 promoter, the siRNA sense and antisenseencoding sequences in the form of a stem-loop, the Pol III terminatorsequence, and the “stuffer” tag sequence (FIG. 1D). To construct thiscassette two 3′ primers are used. The first PCR reaction employs the 5′U6 universal primer and a 3′ primer complementary to 20 nucleotides ofthe U6 promoter, followed by sequences complementary to the siRNA senseencoding sequence and the 9 nt. loop (FIG. 1B). One microliter of thisfirst reaction is reamplified in a second PCR reaction that employs thesame 5′ U6 primer and a 3′ primer harboring sequences complementary tothe 9 nt. loop appended to the antisense strand, Ter and “stuffer” tagsequence (FIG. 1B).

[0065] In another embodiment, a one step PCR reaction is conducted witha single 3′ primer that harbors the sense, loop, antisense, Ter and“stuffer’ tag sequences (FIG. 1C). Although generally effective, thisapproach employs a considerably long and structured 3′ PCR primer thatwith some sequences may cause difficulties in obtaining the desired fulllength, double stranded PCR products.

[0066] PCR conditions are relatively standard for all siRNA genes sincethe regions complementary to the U6 promoter do not change. For theconstruction of several cassettes, optimal amplification was achieved ineach case using 1 minute for each PCR step and 55° C. as annealingtemperature. For direct transfections and testing of the PCR amplifiedsiRNA genes, the 5′ termini of the PCR primers may be modified, forexample, by phosphorylation, preferably using a DNA polynucleotidekinase and non-radioactive ATP. This modification of the primersstabilizes the PCR products intracellularly, thereby enhancing theefficacy of the PCR products.

[0067] Once the PCR reaction is completed, the products can be columnpurified from the primers, e.g., via a gel filtration column or byexcising them directly from a gel following electrophoresis. Thepurified products can be applied to cells following cationic liposomeencapsidation and/or standard transfection procedures, such as thosedescribed below and in co-pending Application Serial No. 60/356,127,filed on Feb. 14, 2002, which is incorporated herein by reference.Intracellular expression of the transfected PCR products was detected byNorthern blotting analyses (FIG. 3A), thus demonstrating goodtransfection efficiency.

[0068] Rapid Screening of Functional siRNAs and Accessible Target SitesUsing siRNA-Encoding PCR Products.

[0069] An HIV rev sequence fused to the enhanced green fluorescentprotein (EGFP)-coding sequence (Lee et al., 2002) was used to test thePCR amplified siRNA encoding DNA for efficacy in cells. This constructwas inserted in the Ecdysone-inducible pIND vector system (Invitrogen).The vector was then transfected into 293 cells, which stably express thetrans-activator for the inducible promoter. Use of this system resultsin strong EGFP expression following addition of Ponasterone A(Invitrogen) to the culture media (FIG. 3A).

[0070] A stable cell line expressing both the trans-activator and targetconstructs may be preferable when multiple siRNA genes are being tested,but co-transfection with the target-EGFP fusion construct provides arapid and sensitive test for siRNA efficacy. Target sequence cDNAs canbe readily cloned into this inducible vector system to create thedesired EGFP fusion. Utilizing this system, an effective siRNA expressedfrom the PCR product will inhibit EGFP expression, allowing either FACSor microscopic based analyses of siRNA function.

[0071] To test the PCR approach, U6 cassettes containing either sense orantisense siRNA genes (FIG. 1A) or a hairpin construct encoding both thesense and antisense si-RNAs (FIG. 1C) were amplified. The PCR productswere column purified. The purified PCR products were then co-transfectedwith the inducible rev-EGFP fusion construct into the Ecdysonetransactivator expressing cell line. 48 hours post transfection,Ponasterone A was added to the culture to induce target mRNA expression.Using this system a strong and specific down regulation of EGFPexpression by the siRNAs was detectable 12 hours post induction (FIG.2). Transfection of a control cassette, such as a U6 expression cassetteexpressing only the sense (FIG. 2B), the antisense (FIG. 2C) or anirrelevant siRNA (not shown), had no effect on expression of EFGP.However, when cassettes expressing the sense and antisense siRNAs wereco-transfected with the target, or when a single cassette containing thehairpin siRNA gene was used, a specific and effective down regulation ofthe target was detected (FIGS. 2D and E). The best and most reproducibleinhibition (nearly 90%) was obtained with the hairpin siRNA expressingcassette. These results were reproduced independently 5 times. Theselected length and sequence of the 9 base loop (UUUGUGUAG) used forthese experiments is based upon phylogenetic comparisons of loops foundin several micro-RNA precursors. When using the above loop, the sequenceof the siRNA sense strand preferably should not include a U as the 3′base since this would create a stretch of 4 Uridines, which can serve asa Pol III terminator element.

[0072] The above results indicate that the transfection-PCR methodologyof the present invention can be easily used to rapidly test siRNAtargeting and function in cells.

[0073] An important element in the design of effective siRNAs is theselection of siRNA/target sequence combinations that yield the bestinhibitory activity. This can be accomplished using siRNAs andtransfection procedures, but this can be costly and time consuming. Byutilizing the PCR strategy, several siRNA genes can be simultaneouslytested in a single transfection experiment.

[0074] In order to facilitate the identification of functional siRNAgenes, a “stuffer” tag sequence was inserted directly after the Pol IIItranscription terminator (see FIG. 1). By utilizing this tag, atransfected PCR cassette can be amplified from transfected cells and thesiRNA sequence subsequently identified (FIG. 1D). This can beaccomplished by utilizing the 5′ U6 universal primer and a primercomplementary to the tag sequence (FIG. 1D). The tag sequence can startwith the 6 Ts of the Ter sequence followed by a restriction site thatcan be used for subsequent cloning, and a “stuffer” of 6 extranucleotides (for a total of 18 nt). A mix of several siRNAs can besimultaneously co-transfected with the inducible target-EGFP cassetteinto the cell line containing the trans-activator. Twelve hours afteradding Ponasterone A, the EGFP negative and EGFP positive cells can becollected by FACS sorting, and the DNAs harvested from both fractions.The isolated PCR products can then be transfected for a second round ofselection and amplification to select those siDNA genes that express themost potent siRNAs. The resultant PCR products can then be cloned andsequenced. The functional siRNA can be identified since it would beabsent in the cells still expressing EGFP but present in the EGFPnegative fraction.

[0075] Using variations of the above approach, several expressioncassettes may be created and used to simultaneously screen for siRNAsensitive target sites on any given mRNA. The target sequence may befused to EFGP or a similar reporter, and screening can be rapidlyaccomplished via FACS analyses and sorting. This strategy can beutilized for endogenous targets when there is a positive selection or aFACS sortable phenotype available. An amplification strategy inaccordance with the present invention offers a rapid and inexpensiveapproach for intracellular expression of siRNAs and subsequent testingof target site sensitivity to down-regulation by siRNAs.

[0076] The present invention is further detailed in the followingExamples, which are offered by way of illustration and are not intendedto limit the invention in any manner.

EXAMPLE 1

[0077] Target Construction and Location of the siRNA Target Site

[0078] The HIV-rev sequence followed by the EGFP gene cloned in thepIND-inducible vector (Invitrogen) as previously described (Lee et al.,2002) was selected as a target site for siRNA. The selection of theaccessible target site for the siRNA was based on previous work and wasshown to be an effective siRNA target using the U6 expression system(Lee et al., 2002). The sequence of the target site is: 5′GCCTGTGCCTCTTCAGCTACC 3′ [SEQ ID NO: 1], which is located 213nucleotides downstream of the rev-AUG start codon.

EXAMPLE 2

[0079] Polymerase Chain Reaction

[0080] PCR reactions were performed using a plasmid containing the humanU6 promoter as template. The 5′ oligonucleotide (5′U6 universal primer)is complementary to 29 nucleotides at the 5′ end of the U6 promoter(bold italics) 5′ATCGCAGATCTGGATCCAAGGTCGGGCAGGAAGAGGGCCT3′ [SEQ ID NO:2] and was used for all PCR steps. The 3′ oligonucleotides, whichcontain either the sense, antisense, or both sense and antisense, aredepicted in FIG. 1 and described herein. The last 20 nucleotides at the3′ end of all 3′ PCR primers are complementary to the last 20nucleotides of the U6 promoter which is: 5′GTGGAAAGGACGAAACACCG3′ [SEQID NO: 3]. All PCR reactions were carried out as follows: 1 min. at 94°C., 1 min at 55° C. and 1 min at 72° C. for 30 cycles. The PCR primerswere kinased with non-radioactive ATP prior to amplification andpurified on Qiagen columns prior to using them in the PCR reactions. ThePCR products were also purified on Quiagen columns.

[0081] The sequences of the siDNA encoding oligos are: 1. Sense forsiRNA rev-5′CGAAAAGGCCTAAAAAAGGTAGCTGAAGAGGCACAGGCGGTGTTTCGTCCTTTCCACAAGATATATAA3′ [SEQ ID NO:4] 2. Antisense for siRNA rev-5′CGAAAAGGCCTAAAAAAGCCTGTGCCTCTTCAGCTACCGGTGTTTCGTCCTTTCCACAAGATATATAA3′ [SEQ ID NO:5] 3. Hairpin siRNA oligo 1- sense5′TACACAAAGGTAGCTGAAGAGGCACAGGCGGTGTTTCGTCCTTTCCACAAGATATATAA 3′ [SEQ IDNO:6] 4. Hairpin siRNA oligo 2-antisense5′CGAAAAGGCCTAAAAAAGCCTGTGCCTCTTCAGCTACCCTACACAAAGG 3′ [SEQ ID NO:7]

[0082] The italicized sequences are the siRNA encoding sequences.

EXAMPLE 3

[0083] Cell Lines and Culture Conditions

[0084] 293 cells were grown DMEM (Irvine Scientific, Santa Ana, Calif.)supplemented with 10% fetal calf serum (Irvine Scientific), 1 mML-glutamine, and 100 units/ml of penicillin/streptomycin. TheEcdysone-inducible stable A293 clone has been previously described (Leeet al., 2002) and it was maintained in DMEM containing 100 μg/ml ofZeocin (Invitrogen).

EXAMPLE 4

[0085] Transfection Conditions for siRNA-PCR Products

[0086] 250 ng of the target plasmid were co-transfected with either: 1)50 ng of the PCR cassette expressing the sense, and/or 50 ng of thecassette expressing the antisense siRNA; or 2) 100 ng of the singlecassette expressing both the sense and antisense linked by a 9 nt. loop.As few as 25 ng of the stem loop siRNA was effective in blocking targetexpression. An irrelevant stem-loop siRNA was used as an additionalcontrol and did not result in any effect on target expression (notshown).

[0087] To facilitate the transfection of the small amounts of PCRamplified DNA, 400 ng of Bluescript plasmid were added to each reactionto serve as carrier. 5 μM Ponasterone A was added to the culture media48 hours after transfection, and the cells were analyzed for EGFPexpression 12 hours following induction. Transfections were performed in6 well plates using Lipofectamine Plus™ reagent (Life Technologies,GibcoBRL) as described by the manufacturer. For microscope imaging,cells were grown and transfected on glass coverslips treated with 0.5%gelatin (Sigma). 12 hours post-induction the coverslips were lifted fromthe 6 well plate and treated for 10 min. at room temperature with 4% PFAfor cell fixation. Cell nuclei were visualized adding DAPI to themounting solution. Down regulation of the rev-EGFP mRNA was quantitatedby FACS analyses.

EXAMPLE 5

[0088] Northern Analyses

[0089] Total RNA was isolated using RNA STAT-60 (TEL-TEST B Inc.,Friendswood, Tex.) according to the manufacturer's instructions. 5 μg oftotal RNA was fractionated by 8M-6% PAGE, and transferred onto Hybond-N+membrane (Amersham Pharmacia Biotech). A ³²P-radiolabeled 21-mer probecomplementary to the si-antisense RNA was used for the hybridizationreactions, which were performed for 16 hours at 37° C. A 21-mer DNAoligonucleotide was electrophoresed alongside the RNA samples and usedas size and hybridization control (not shown).

EXAMPLE 6

[0090] Direct Amplification of siRNAs from Cell Lysates

[0091] EGFP-negative and -positive cell fractions were collected by FACSsorting. The cell pellets were recovered immediately by centrifugationof the sorted fractions. The pellets were lysed in 50 mM KCl, 10mMTris-HCl (pH 8.0), 1.25 mM MgCl₂, 0.45% NP40, 0.45% Tween, and 0.75μg/μl Proteinase K at 37° C. overnight. After 10 minutes heatinactivation at 95° C., 3 μl of the cell lysates were used directly inPCR reactions.

EXAMPLE 7

[0092] 15 ng of the PCR amplified gene encoding the siRNA hairpintargeting the HIV-rev site, along with 15 ng of an irrelevant siRNA PCRproduct were co-transfected with the inducible target-EGFP cassette intothe 293 cell line expressing the trans-activator. Twelve hours posttransfection, Ponasterone A was added to induce EGFP expression and theEGFP negative and positive cells were FACS sorted. The cell pellets fromboth the EGFP negative and positive cells were collected bycentrifugation, lysed overnight in lysis buffer and the DNAs amplifieddirectly by PCR utilizing the appropriate primer sets. Two different 3′primers that discriminate between the two different siRNA encoding DNAcassettes were used. It was expected that the non-functional siRNAexpression cassette should be detectable by PCR amplification in bothcell fractions, whereas the functional siRNA expression cassette wouldonly be detectable in the EFGP negative fraction since its productswould have functionally downregulated EGFP expression. The results oftwo independent experiments are shown in FIGS. 3B and C. In both cases,the non-functional siRNA encoding gene was PCR amplified from allfractions (FIG. 3B), whereas the functional siRNA encoding expressionconstruct was primarily detected in the EGFP negative cell fractions(FIG. 3B).

EXAMPLE 8

[0093] Anti-HIV U6+1 short hairpin siRNA (shRNA) PCR products wereproduced by PCR using a U6+1 promoter construct as template (pTZU6+1), auniversal 5′ primer, and a specific 3′ primer. The primers are shown inTable 1 in the standard 5′ to 3′ orientation. The shRNAs were designedto be transcribed in the sense target sequence-loop-antisense targetsequence-UUUUU (pol3 terminator) format. The sequence of the universal5′ primer, which anneals to the 5′ end of the U6+1 promoter, also isshown in Table 1. Table 1 further shows the corresponding sequence ofthe 3′ end of expected PCR product (the upper, coding strand is shown inthe standard 5′ to 3′ orientation), beginning with the 3′ end of theU6+1 promoter, ending with the +1 start site of transcription, followedby the sequences coding for the hairpin RNA (sensetarget/loop/anti-sense), the Pol III terminator, Bgl2 site and extranucleotides. The sequences of the 3′ primers also are shown in Table 1following the sequence of each PCR product.

[0094] Table 1 also shows a SELEX 2144 tRNA^(Lys3)-tat/rev target21-stem shRNA.

EXAMPLE 9

[0095] PCR-amplified expression cassettes expressing anti-tat siRNA werefound to potently inhibit HIV infection. PCR amplified short hairpin RNAencoding genes U6+1NLS1(tat/rev)shRNA, versus the same gene in a plasmidvector (pBS U6+1 NLS1 (tat/rev)shRNA), in the amounts indicated in FIG.4, were co-transfected with 0.5 micrograms of HIV pNL4-3 into 293 cellsand the viral encoded p24 antigen output was measured over three days.As controls, empty vector backbones (pBS or pTZU6+1) or a triplemutation at sites 9, 10 and 11 of the anti-tat shRNA (U6+1mNLS1shRNA(PCR product) or pBSU6+1mNLS1shRNA (plasmid based system)) weretransfected as a PCR amplified gene with HIV-1. The results in FIG. 4show the several logs worth of inhibition obtained using cassettesproduced according to the present invention as compared to the same genein a plasmid vector.

[0096]FIG. 4 shows that the U6+1 tat/rev shRNA specifically mediatescomparable HIV inhibition at equivalent molar ratios, regardless ofwhether the cassette is transfected as a PCR product directly or is partof a plasmid backbone. This panel also illustrates that small amounts ofan shRNA construct can mediate substantial HIV inhibition withsusceptible target sites.

EXAMPLE 10

[0097]FIG. 5 shows the persistence of HIV inhibition by shRNAs expressedfrom PCR products. HIV co-transfection inhibition assays were performedas described in Example 9, using the U6+1 tat/rev shoRNA U6+1 tat/revmutant shRNA constructs as positive and negative controls. Aftercollecting viral supernatant on day 3, the confluent 293 cells werereseeded in fresh medium at 10% confluency and allowed to expand foranother 3 days before collecting viral supernatants for assay. FIG. 5shows that PCR product-mediated HIV inhibition persists for at least sixdays under these experimental conditions.

EXAMPLE 11

[0098]FIG. 6 shows the results of an HIV inhibition test using clonedU6+1 env shRNA constructs (Table 1). Each well of a 6-cluster platecontaining 293 cells at ˜50% confluency was co-transfected with 0.5 μgof cloned plasmid shRNA and 0.5 μg pNL4-3 proviral DNA per well of a6-cluster plate using Lipfectamine Plus according to the manufacturer'sinstructions. Aliquots of viral supernatants were taken at the indicatedtimes and assayed for p24 antigen. Tat/rev, positive control forinhibition; Mtat/rev, negative control for inhibition mismatched withtarget site at positions 10, 11, and 12 relative to 5′ end of processedantisense strand.

EXAMPLE 12

[0099]FIG. 7 shows the results of HIV inihibition assays, using 200 ngof each U6+1 env shRNA PCR product purified from a set of PCR reactions.pTZU6+1 is a negative control plasmid containing the U6+1 promoter.

EXAMPLE 13

[0100]FIG. 8 shows HIV inhibition by U6+1 env shRNAs, using 100 ng eachU6+1 env shRNA PCR product purified from another set of PCR reactions.U6+1 tat/rev shRNA and U6+1 tat/rev Mutant shRNA PCR products wereincluded as controls.

[0101] The publications and other materials used herein to illuminatethe background of the invention, and provide additional detailsrespecting the practice of the invention, are incorporated herein byreference as if each was individually incorporated herein by reference.

[0102] While the invention has been disclosed in this patent applicationby reference to the details of preferred embodiments of the invention,it is to be understood that the disclosure is intended in anillustrative rather than in a limiting sense, as it is contemplated thatmodifications will readily occur to those skilled in the art, within thespirit of the invention and the scope of the appended claims.

REFERENCES

[0103] 1 Moss, E. G., RNA interference: it's a small RNA world. CurrBiol, 2001. 11(19): p. R772-5.

[0104] 2. Bernstein, E., A. M. Denli, and G. J. Hannon, The rest issilence. Rna, 2001. 7(11): p. 1509-21.

[0105] 3. Elbashir, S. M., W. Lendeckel, and T. Tuschl, RNA interferenceis mediated by 21- and 22-nucleotide RNAs. Genes Dev, 2001. 15(2): p.188-200.

[0106] 4. Elbashir, S. M., et al., Duplexes of 21-nucleotide RNAsmediate RNA interference in cultured mammalian cells. Nature, 2001.411(6836): p. 494-8.

[0107] 5. Bernstein, E., et al., Role for a bidentate ribonuclease inthe initiation step of RNA interference. Nature, 2001. 409(6818): p.363-6.

[0108] 6. Hammond, S. M., et al., Argonaute2, a link between genetic andbiochemical analyses of RNAi. Science, 2001. 293(5532): p. 1146-50.

[0109] 7. Lee, N. S., et al., Expression of small interfering RNAstargeted against HIV-1 rev transcripts in human cells. Nat Biotechnol,2002. 20(5): p. 500-5.

[0110] 8. Miyagishi, M. and K. Taira, U6 promoter driven siRNAs withfour uridine 3′ overhangs efficiently suppress targeted gene expressionin mammalian cells. Nat Biotechnol, 2002. 20(5): p. 497-500.

[0111] 9. Paul, C. P., et al., Effective expression of small interferingRNA in human cells. Nat Biotechnol, 2002. 20(5): p. 505-8.

[0112] 10. Brummelkamp, T. R., R. Bernards, and R. Agami, A system forstable expression of short interfering RNAs in mammalian cells. Science,2002. 296(5567): p. 550-3.

[0113] 11. Ketting, R. F., et al., Dicer functions of RNA interferenceand in synthesis of small RNA involved in developmental timing in C.elegans. Genes Dev, 2001. 15(20): p. 2654-9.

[0114] 12. Yu, J. Y., S. L. DeRuiter, and D. L. Turner, RNA interferenceby expression of short-interfering RNAs and hairpin RNAs in mammaliancells. Proc Natl Acad Sci USA, 2002. 99(9): p. 6047-52.

[0115] 13. Holen, T., et al., Positional effects of short interferingRNAs targeting the human coagulation trigger Tissue Factor. NucleicAcids Res, 2002. 30(8): p. 1757-66.

[0116] 14. Hamilton, A., et al., Two classes of short interfering RNA inRNA silencing. EMBO Journal, 2002. 21: p. 4671-4679.

[0117] 15. Herman, J. G., et al., Methylation-Specific PCR: A Novel PCRAssay for Methylation Status of CpG Islands. PNAS USA, 1996. 93: p.9821-9826.

1 38 1 21 DNA Artificial Sequence chimeric nucleotide target sequence 1gcctgtgcct cttcagctac c 21 2 40 DNA Artificial Sequence oligonucleotideprimer 2 atcgcagatc tggatccaag gtcgggcagg aagagggcct 40 3 20 DNA Homosapiens misc_feature (1)..(20) homologous to the 3′ end of the u6promoter 3 gtggaaagga cgaaacaccg 20 4 68 DNA Human immunodeficiencyvirus misc_feature (18)..(38) siRNA encoding sequence 4 cgaaaaggcctaaaaaaggt agctgaagag gcacaggcgg tgtttcgtcc tttccacaag 60 atatataa 68 568 DNA Human immunodeficiency virus misc_feature (18)..(38) siRNAencoding sequence 5 cgaaaaggcc taaaaaagcc tgtgcctctt cagctaccggtgtttcgtcc tttccacaag 60 atatataa 68 6 58 DNA Human immunodeficiencyvirus misc_feature (9)..(29) siRNA encoding sequence 6 tacacaaaggtagctgaaga ggcacaggcg gtgtttcgtc ctttccacaa gatatata 58 7 49 DNA Humanimmunodeficiency virus misc_feature (18)..(38) siRNA encoding sequence 7cgaaaaggcc taaaaaagcc tgtgcctctt cagctaccct acacaaagg 49 8 36 DNAArtificial Sequence chimeric nucleotide construct 8 ataagaatgcggccgccccg gggatccaag gtcggg 36 9 89 DNA Artificial Sequence chimericnucleotide construct 9 cttgtggaaa ggacgaaaca ccgcaacaca actgtttaatagtatttgtg tagtactatt 60 aaacagttgt gttgtttttt agatcttcc 89 10 89 DNAArtificial Sequence chimeric nucleotide construct 10 ggaagatctaaaaaacaaca caactgttta atagtactac acaaatacta ttaaacagtt 60 gtgttgcggtgtttcgtcct ttccacaag 89 11 89 DNA Artificial Sequence chimericnucleotide construct 11 cttgtggaaa ggacgaaaca ccgcacaatc acactcccatgcagtttgtg tagctgcatg 60 ggagtgtgat tgtgtttttt agatcttcc 89 12 89 DNAArtificial Sequence chimeric nucleotide construct 12 ggaagatctaaaaaacacaa tcacactccc atgcagctac acaaactgca tgggagtgtg 60 attgtgcggtgtttcgtcct ttccacaag 89 13 88 DNA Artificial Sequence chimericnucleotide construct 13 cttgtggaaa ggacgaaaca ccggaggagg cgatatgagggactttgtgt aggtccctca 60 tatcgcctcc tcctttttta gatcttcc 88 14 88 DNAArtificial Sequence chimeric oligonucleotide construct 14 ggaagatctaaaaaaggagg aggcgatatg agggacctac acaaagtccc tcatatcgcc 60 tcctccggtgtttcgtcctt tccacaag 88 15 89 DNA Artificial Sequence chimericoligonucleotide construct 15 cttgtggaaa ggacgaaaca ccgtgtctga tatagtgcagcagctttgtg taggctgctg 60 cactatatca gacatttttt agatcttcc 89 16 89 DNAArtificial Sequence chimeric nucleotide construct 16 ggaagatctaaaaaatgtct gatatagtgc agcagcctac acaaagctgc tgcactatat 60 cagacacggtgtttcgtcct ttccacaag 89 17 89 DNA Artificial Sequence chimericnucleotide construct 17 cttgtggaaa ggacgaaaca ccgtctgttg caactcacagtctgtttgtg tagcagactg 60 tgagttgcaa cagatttttt agatcttcc 89 18 89 DNAArtificial Sequence chimeric oligonucleotide construct 18 ggaagatctaaaaaatctgt tgcaactcac agtctgctac acaaacagac tgtgagttgc 60 aacagacggtgtttcgtcct ttccacaag 89 19 88 DNA Artificial Sequence chimericnucleotide construct 19 cttgtggaaa ggacgaaaca ccgcggagac agcgacgaagagctttgtgt aggctcttcg 60 tcgctgtctc cgctttttta gatcttcc 88 20 88 DNAArtificial Sequence chimeric nucleotide construct 20 ggaagatctaaaaaagcgga gacagcgacg aagagcctac acaaagctct tcgtcgctgt 60 ctccgcggtgtttcgtcctt tccacaag 88 21 88 DNA Artificial Sequence chimeric nucleotideconstruct 21 cttgtggaaa ggacgaaaca ccgcggagac atatacgaag agctttgtgtaggctcttcg 60 tatatgtctc cgctttttta gatcttcc 88 22 88 DNA ArtificialSequence chimeric oligonucleotide construct 22 ggaagatcta aaaaagcggagacatatacg aagagcctac acaaagctct tcgtatatgt 60 ctccgcggtg tttcgtcctttccacaag 88 23 88 DNA Artificial Sequence chimeric oligonucleotideconstruct 23 cttgtggaaa ggacgaaaca ccgcggagac agcgacgaag agctttgtacaggctcttcg 60 tcgctgtctc cgctttttta gatcttcc 88 24 88 DNA ArtificialSequence chimeric nucleotide construct 24 ggaagatcta aaaaagcggagacagcgacg aagagcctgt acaaagctct tcgtcgctgt 60 ctccgcggtg tttcgtcctttccacaag 88 25 14 DNA Human immunodeficiency virus 25 ttccagtcag acct 1426 27 DNA Artificial Sequence oligonucleotide 26 ttttccagtc acacctcaggtaccttt 27 27 91 DNA Artificial Sequence chimeric nucleotide construct27 cttgtggaaa ggacgaaaca ccgttccagt cacacctcag gtactttgtg taggtacctg 60aggtgtgact ggaatttttt agatcttaac c 91 28 91 DNA Artificial Sequencechimeric nucleotide construct 28 ggttaagatc taaaaaattc cagtcacacctcaggtacct acacaaagta cctgaggtgt 60 gactggaacg gtgtttcgtc ctttccacaa g91 29 19 DNA Artificial Sequence oligonucleotide 29 gctctattag atacaggag19 30 27 DNA Artificial Sequence oligonucleotide 30 gaagctctattagatacagg agcagat 27 31 90 DNA Artificial Sequence chimeric nucleotideconstruct 31 cttgtggaaa ggacgaaaca ccgctctatt agatacagga gcatttgtgtagtgctcctg 60 tatctaatag agctttttta gatcttaacc 90 32 90 DNA ArtificialSequence chimeric nucleotide construct 32 ggttaagatc taaaaaagctctattagata caggagcact acacaaatgc tcctgtatct 60 aatagagcgg tgtttcgtcctttccacaag 90 33 100 DNA Artificial Sequence chimeric nucleotideconstruct 33 cttgtggaaa ggacgaaaca ccgcctgtgc ctcttcagct accgaagcttgggtagctga 60 agaggcacag gcttttttca tgcatgcatg tcccggggga 100 34 98 DNAArtificial Sequence chimeric nucleotide construct 34 acacctttcctgctttgtgg cggacacgga gaagtcgatg gcttcgaacc catcgacttc 60 tccgtgtccgaaaaaagtac gtacgtacag ggccccct 98 35 29 DNA Artificial Sequence primer35 acgcgtcgac gcccggatag ctcggtcgg 29 36 82 DNA Artificial Sequencechimeric nucletide construct 36 gtcgacgccc ggatagctcn gtcggtngagcatcagactt ttaatctgag ggtccagggt 60 tcnagtccct gttcgngcnc ca 82 37 90DNA Artificial Sequence chimeric nucleotide construct 37 gttcgagtccctgttcgtgc accagcggag acagcgacga agagctttgt gtaggctctt 60 cgtcgctgtctccgcttttt tagatcttcc 90 38 90 DNA Artificial Sequence chimericnucleotide construct 38 ggaagatcta aaaaagcgga gacagcgacg aagagcctacacaaagctct tcgtcgctgt 60 ctccgcgctc agggacaagc acgtggtaac 90

What is claimed is:
 1. An amplification-based method for producing apromoter-containing siRNA expression cassette, comprising: i) treatingone strand of a double-stranded promoter sequence, in an amplificationreaction mixture, with an oligonucleotide primer which is complementaryto the 5′ end of the promoter sequence; ii) treating the other strand ofthe promoter sequence, in the amplification reaction mixture, with asecond oligonucleotide primer which is complementary to the 3′ end ofthe promoter sequence, wherein the second primer comprises one or moresequences which are complementary to a sequence encoding a sense and/orantisense sequence of a siRNA molecule, along with one or both of a loopsequence and a terminator sequence; and iii) treating the amplificationreaction mixture of steps (i) and (ii) in an amplification reaction at atemperature for annealing and extending said primers on the promotersequence and at a temperature for denaturing the extension products toprovide an amplified product comprising the promoter, one or moresequences encoding the sense and/or antisense sequence of the siRNAmolecule, and one or both of the loop sequence and the terminatorsequence, and wherein steps (i)-(iii) are repeated a sufficient numberof times to amplify the promoter-containing siRNA expression cassette.2. The method of claim 1, wherein the method is a PCR-based method. 3.The method of claim 1, wherein the promoter is a Pol III promoter. 4.The method of claim 3, wherein the Pol III promoter is a mammalian U6promoter.
 5. The method of claim 4, wherein the U6 promoter is a humanU6 promoter.
 6. The method of claim 1, wherein the sequence encoding theterminator sequence comprises a sequence of about 4-6 deoxyadenosines.7. The method of claim 6, wherein the sequence encoding the terminatorsequence comprises a sequence of 6 deoxyadenosines.
 8. The method ofclaim 1, wherein the second primer further comprises a tag sequence toidentify functional siRNA encoding sequences.
 9. The method of claim 8,wherein the tag sequence further comprises a restriction site useful forcloning.
 10. The method of claim 1, wherein the second primer comprisesa sequence that is complementary to a sequence encoding a sense sequenceand a sequence that is complementary to a sequence encoding an antisensesequence of said siRNA molecule, along with a terminator sequence. 11.The method of claim 12, wherein the sequences complementary to asequence encoding the sense and antisense sequences are attached by aloop sequence.
 12. The method of claim 13, wherein the loop sequencecontains about 6 to about 9 nucleotides.
 13. The method of claim 1,wherein the amplified product comprises the promoter sequence, asequence encoding either the sense or antisense sequence of the siRNAmolecule, and the loop sequence or the terminator sequence.
 14. Themethod of claim 13, wherein the amplified product comprises the promotersequence, a sequence encoding either the sense or antisense sequence ofthe siRNA molecule, and the terminator sequence.
 15. The method of claim13, wherein the amplified product comprises the promoter sequence, asequence encoding either the sense or antisense sequence of the siRNAmolecule, and the loop sequence, said method further comprising the stepof treating the amplified product, in another amplification reaction,with a third oligonucleotide primer, a portion of which is complementaryto the loop sequence of the first amplified product, and which comprisesa sequence complementary to a sequence encoding the antisense sequencewhen the first amplified product contains the sense encoding sequence,or a sequence complementary to a sequence encoding the sense sequencewhen the first amplified product contains the antisense encodingsequence, along with a terminator sequence, to provide a secondamplified product.
 16. The method of claim 15, wherein the secondamplified product comprises the promoter sequence, a sequence encodingthe sense sequence and a sequence encoding the antisense sequence of thesiRNA molecule, wherein the sense and antisense sequences are attachedby a loop sequence, and the terminator sequence.
 17. The method of claim1, further comprising the step of transfecting a cell with the amplifiedpromoter-containing siRNA expression cassette, wherein an siRNA moleculeis expressed.
 18. The method of claim 17, wherein the selected cells aremammalian cells.
 19. The method of claim 17, wherein one or more of theoligonucleotide primers are modified.
 20. The method of claim 19,wherein one or more of the oligonucleotide primers are modified byphosphorylation.
 21. The method of claim 17, further comprising the stepof screening for a target site on mRNA sensitive to the expressed siRNAmolecule.
 22. The method of claim 17, wherein the cell is transfectedwith two or more different siRNA expression cassettes.
 23. The method ofclaim 22, wherein the different siRNA expression cassettes contain oneor both of a different siRNA encoding gene and a different promoter. 24.A PCR-based approach in the form of a kit for producing apromoter-containing siRNA expression cassette, comprising adouble-stranded, promoter-containing template, an oligonucleotide primercomplementary to the 5′ end of the promoter-containing template, and anoligonucleotide primer complementary to the 3′ end of thepromoter-containing template, wherein the 3′ primer comprises one ormore sequences complementary to a sequence encoding a sense and/orantisense sequence of a siRNA or siRNA molecule.
 25. The PCR-basedapproach of claim 24, wherein the promoter is a Pol III promoter. 26.The PCR-based approach of claim 25, wherein the Pol III promoter ismammalian U6 promoter.
 27. The PCR-based method of claim 26, wherein theU6 promoter is a human U6 promoter.
 28. A method for screening potentialtarget sequences susceptible to siRNA mediated degradation, comprisingtransfecting a cell with an amplified siRNA expression cassette underconditions in which an siRNA molecule can be expressed and mediatedegradation of the potential target sequences.
 29. A method forinhibiting expression of a target gene, comprising transfecting a cellwith an amplified siRNA expression cassette under conditions in which ansiRNA molecule can be expressed and inhibit expression of the targetgene.